N-type (CaV2.2) voltage-gated calcium channels (VGCCs) are expressed at presynaptic terminals in virtually all central and peripheral neurons where they control the calcium entry that triggers exocytosis. Therefore, the efficacy of neurotransmission is directly correlated with the amount of calcium that passes through VGCCs. This relationship is exploited by a classic form of presynaptic regulation: G protein-mediated inhibition of N-type channels. The molecular rules governing the specific coupling of G protein-coupled receptors (GPCRs) to calcium channels are not fully understood. Recent work from the Lipscombe lab suggests an exciting and novel explanation for channel-G protein interaction specificity: alternative splicing of CaV2.2 pre-mRNA is the molecular mechanism that controls G protein coupling to the N-type calcium channel. This proposal focuses on the role of an alternative exon, el 8a, in the G protein-mediated inhibition of the N-type channel. The overarching hypothesis of this proposal is: cell- specific inclusion of el 8a renders N-type channels susceptible voltage-independent inhibition by GPCRs. The specific aims of this project are to: (1) determine which G proteins couple to e18a;(2) determine how el 8a contributes to the inhibition of the N-type channel by the D1 dopamine receptor (D1R) and the Angiotensin II receptor type 1 (AT1R);(3) determine the role of el8a in mediating G protein-dependent inhibition of native N-type currents in dopaminergic neurons. N-type channel el 8a splice variants will be studied both in an expression system (tsA201 cells) and in dissociated neurons from mouse midbrain cultures engineered to express GFP in DIR-expressing neurons. The function of the channels will be probed using electrophysiology and pharmacology. G protein inhibition of cloned and native channels will be assessed with GTPgS or GPCR agonists. This inhibition will be disrupted by (1) specifically inhibiting N-type channels containing e18a with siRNAs, and (2) sequestering el 8a binding partners with an inhibitory peptide. Overall, this project serves to elucidate the role of alternative splicing of the N-type channel in controlling susceptibility to G protein-mediated inhibition. RELEVANCE: Neurodegeneration in Parkinson's Disease specifically affects midbrain neurons. Neurotransmitters can modulate the activity of these neurons by inhibiting calcium channels, proteins vital to communication between neurons. This research project serves to understand the inhibition of calcium channels specifically expressed in midbrain neurons.